Antithrombin-III (AT-III) is an .alpha..sub.2 globulin known to inhibit the coagulation of blood. AT-III acts essentially as an irreversible inhibitor. It is believed that AT-III inhibits serine proteases in plasma during the activation of either the coagulation or the fibrinolytic systems. AT-III has significant inhibitory activity of factor Xa and thrombin.
Families congenitally deficient in AT-III have a high incidence of thromboses. Administration of AT-III to patients with thrombotic disorders has been attempted.
There are reports of attempts to purify AT-III from human plasma and plasma pastes using conventional techniques. However, those procedures were lengthy or the yields were poor. A substantial improvement in the purification methodology took place with the incorporation of an affinity chromatography step using purified heparin as the solid phase bound ligand.
Damus & Wallace (Biochem. Biophys. Res. Comm., 61 (4); 1147, 1974) purified canine AT-III in a scheme that incorporated heparin-Sepharose chromatography. Heat defibrinated plasma was passed over a small column containing heparin-Sepharose at 8.degree. C. The end product was still heterogeneous as determined by the distribution of specific activity throughout the elution profile.
Miller-Andersson et al. (Thromb. Res., 5:439, 1974) teaches the use of heparin-Sepharose to purify human AT-III. Citrated human plasma that had been frozen immediately after removal of blood cells by centrifugation was added in batch fashion to heparin-Sepharose at 5.degree. C. The gel suspension then was poured into a column, allowed to settle and the adsorbed material was eluted with a salt gradient. The entire procedure, which included ion exchange and gel filtration chromatography, provided a 34% yield. The large number of chromatographic separations minimizes the possibility of large scale production of AT-III by that method.
Thaler & Schmer (Br. J. Haemat., 31:233, 1975) described an isolation procedure for human and bovine AT-III that involved heparin-agarose chromatography and polyethylene glycol precipitation. All procedures were carried out at 4.degree. C. Either chromatography or precipitation can serve as the initial step in the purification scheme.
Wickerhauser et al. (Vox Sang., 36:281, 1979) describes a large scale method for the preparation of AT-III from plasma or from Cohn fraction IV-VI. The method begins with a polyethylene glycol precipitation followed by batch adsorption on heparin-Sepharose, desalting by ultrafiltration and followed by pasteurization of the final product. The recovery by activity was 32% from plasma and 16% from Cohn fraction IV. All purification steps were carried out at 5.degree. C.